Mutations in either type 1 ryanodine receptor (RyR1) or the dihydropyridine receptor subunit Cav1.1 cause malignant hyperthermia susceptibility (MHS) in humans and animals. Project 2 will address key molecular events by which MHS mutations alter basal RyR1 Ca2+ channel and leak and confer sensitivity to triggering agents. It is necessary to also understand how altered RyR1 channel regulation influences basal changes in mitochondrial bioenergetics, produces oxidative stress, and promotes progressive muscle damage. Four interelated hypotheses are addressed in skeletal muscle and muscle cells obtained from mice and human biopsies to understand how MHS mutations differentially alter (1) the biochemical and biophysical properties of RyR1 channel regulation and their underlying posttranslational modifications, (2) adaptive changes in mitochondrial bioenergetics and whole animal energy expenditure, and (3) if RyR1 channel and mitochondrial dysfunctions can be mitigated or abrogated by molecular and pharmacological interventions that reduce RyR1 leak, abusive Ca2+ entry, increase SR Ca2+ load or specifically scavenge the reactive oxidized lipid product ketoaldehyde (yKA). How RyR1 channel regulation by cytoplasmic/luminal Ca2+, Mg2+ and glutathione redox potential differ among MHS mutations as a result of posttranslational modifications of RyR1 (phosphorylation, nitrosylation, and formation of Lys-lactam adducts) will be investigated in four knock-in mouse models and human muscle biopsies with known MHS mutations. We will investigate the links between RyR1 dysfunction, mitochondrial matrix Ca2+, ROS production, mtDNA copy number and adaptations in Complex activities. Oxygen consumption and acidification rates will be investigated in mouse and human muscle cells under basal and after exposure to volatile anesthetics conditions. Whole body calorimetry will be used to measure resting energy expenditure and nutrient utilization rates of WT and MHS mice and how these parameters are influenced by ambient temperature, fasting and glucose challenge. The proposed studies are transformative because they will lead to understanding how MHS mutations produce phenotypic differences in clinical MH and progressive muscle damage, and test novel intervention strategies to mitigate these interrelated processes.